Systemic mycoses are very important infections in immunocompromised patients. In granulocytopenic patients following chemotherapy, bone marrow or organ transplant, Candida, Aspergillus and Mucoraceae cause serious fungal infections with current mortality rates of about 40% despite of the empirical anti-fungal therapy. In AIDS patients, Histoplasma for North America, Penicillium for Southeast Asia and Crytococcus occur most frequently. The mortality rate is usually very high.
The high mortality rate is partly due to the inability of making early, specific diagnosis of systemic fungal infections and therefore, the antifungal therapy is often delayed. Because of the low efficacy and high toxicity of antifungal drugs, there has been a significant limitation of prophylaxis with antifungal drugs (for review, J. L. Wheat, Clinical Approach to Infection in the Compromised Host, 3rd ed. Planum Medical Book Company, New York, 1994).
There has been a lack of specific test for the diagnosis of systemic fungal infections. Most of the fungal infections can not be easily diagnosed. With the exception of Cryptococcus that depends on the detection of fungal polysaccharide antigen in serum and Histoplasma in a crude cell extract (histoplasmin), the serology and antigen tests are virtually not effective in the diagnosis of systemic fungal infection due to the lack of sensitive and specificity.
Serology and antigen tests are most frequently used for the diagnosis of viral infections such as HIV and viral hepatitis. In all these cases, recombinant viral proteins and their antibodies are used for the tests. For most systemic fungal infections, however, the laboratory tests for diagnosis are usually unsatisfactory. Essentially all tests are made of crude antigens such as polysaccharides, therefore lack the desired specificity in most cases. Specific recombinant protein based diagnosis can be highly specific. Unlike viruses, however, pathogenic fungi are of far greater genomic complexity and most of the fungal genes can not be used for diagnosis. Therefore, to identify the very few good genes, if any, become the essence of this invention.
At present, the only fungal protein has been demonstrated to have very limited value in the diagnosis of the systemic fungal infection is Candida enolase (Walsh et al., New England Journal of Medicine, 324, 1026-1031). An enzyme assay was established for antigenaemia of enolase for the diagnosis of deep infection by Candida, however, there has been significant limitation in the sensitive of the assay. The application of such a test is very limited since tests on sequential specimens are needed to achieve only very moderate sensitive.
A year ago, it was reported in Lancet (Lancet 1994, 344, 444-5) about the serodiagnosis of Penicillium marneffei systemic infection. In that study, it was observed Penicillium marneffei infection in nearly 10% patients who have persistent fever despite multiple antimicrobials, and in 12% AIDS patients. Similar results were also obtained by Wong et al. at Queen Elizabeth Hospital in Hong Kong (Chung-Hua-I-Hsueh-Tsa-Chih-Taipei 1995, 55,127-36). The conventional method of diagnosis that depends on the isolation of the pathogen from blood specimens was very unsatisfactory, yielding only 10% success. Tissue biopsies were eventually obtained for the confirmation of diagnosis. The mean delay was 11 weeks. To solve the problem, it was established the serodiagnosis assay for Penicillium marneffei infection using immunofluorescent micrograph to determine the antibody titer against the fixed fungal cells. However, this assay requires special skills and equipment to perform. It is lengthy and technically more difficult. Most importantly, it is a very crude assay using whole cells as the antigen and therefore lacks adequate specificity.
Penicillium marneffei is one of the most important fungal pathogen in AIDS patients in Southeast Asia, including Thailand, Indonesia, Hong Kong and China. It was also reported in Europe and North America as imported pathogen carried by travelers. It causes disseminated infection which is always associated with non-specific symptoms including fever, anemia, weight loss and sometimes, skin lesions. In one of the more extensive recent studies done in Thailand (Lancet 1994, 344,110-3), 20% of AIDS people developed Penicillium marneffei infection. About half of the total cases (92) presented skin lesion which allowed the diagnosis through invasive procedures including bone-marrow aspirate and/or lymphoid and skin biopsies. The diagnosis was often delayed in patients who did not present skin lesions. Most patients who were diagnosed early responded to anti-fungal drugs, whereas most who were not diagnosed and treated died. Due to the high toxicity of anti-fungal drugs and the non-specific symptoms of Penicillium marneffei infection, early diagnosis is essential to achieve the cure.